1. Field of the Invention
This invention relates to a novel technique for preparing immunogen conjugates for use in stimulating production of antibodies against a particular hapten. Novel immunogen conjugates and antibodies prepared against such conjugates are provided. Such antibodies are particularly useful as reagents in immunoassays.
Immunoassays are analytical procedures based on specific recognition of the analyte of interest by an appropriately obtained antibody. Antibodies against antigenic analytes are obtained by injecting the antigen into the bloodstream of an animal such as a rabbit. The antigen is recognized as foreign by the immune system of the animal which is accordingly stimulated to produce antibodies to bind the antigen and neutralize it. Serum from such an animal will, therefore, contain immunolobulin proteins (humoral antibodies) which possess a high binding affinity for the antigen inducing the response [Ligand Assay, J. Langan and J. J. Clapp, Eds., Masson Publ. USA, Inc. (New York, 1981), p. 1 et seq]. Substances of relatively low molecular weight, e.g., less than 1500, however, may be only weakly antigenic or unable to stimulate antibody production at all. Nevertheless, antibodies can be raised to such small molecules (referred to as haptens) by immunization with conjugates made up of such low molecular weight substances covalently linked to immunogenic carrier molcules, commonly proteins or polypeptides. The most common protein carriers are the serum albumins of various species, hemocyanin, thyroglobulin and fibrinogen [Methods in Enzymology, Vol. 70, H. Van Vunakis and J. J. Langone, Eds., Academic Press (New York, 1980), p 85].
The type of linkage by which the hapten is attached to the carrier is important for optimum antibody production. Coupling the hapten by means of amide linkages to the terminal amino or carboxyl functions of the protein has been reported to give conjugates of high antigenicity, presumably because this mode of coupling locates the haptens on the surface of the macromolecule where they are more accessible to the receptors in the lymphoid cells [N. Hanna et al, Proc. Soc. Exp. Biol. Med. 140(1): 89-92 (1972)]. Many reagents adapted from peptide chemistry, including carbonyl diimidazole, have been used to attach carboxyl- or amino-functionalized haptens to proteins. These methods are all based on activating the carboxyl component toward amide bond formation [see The Peptides, Vol. I, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1979, p. 66; and U. Axen, Prostaglandins 5(1): 45-7 (1974)]. Difficulties are encountered, however, when this approach is used to link amino-containing haptens to proteins. Proteins contain both amino and carboxyl groups. Activation of protein carboxyl groups, particularly by carbodiimides, tends to form intra and intermolecular amide bonds and polymerize or crosslink the protein, often forming insoluble complexes [S. Bauminger and M. Wilcheck, Methods Enzymol. 70 (Part A): 159 (1980)].
2. Description of the Prior Art
One way reported in the literature to circumvent the crosslinking problem is to attach amino-functionalized haptens to the amino groups of the protein carrier by means of bifunctional reagents which do not activate carboxyl or other functional groups. Many such reagents are known and include toluene-2,4-diisocyanate [C. H. W. Hirs, and S. N. Timasheff, Methods Enzymol. 25 (Part B): 625 (1972)]; difluorodinitrobenzene [H. S. Tager, Anal. Biochem. 71 (2): 367-75 (1976)]; glutaraldehyde [L. A. Frohman et al, Endocrinol. 87: 1055 (1970)]; trichlorotriazine [T. Lang et al, J. C. S. Perkin I: 2889 (1977)]; 4-fluoro-3-nitrophenyl sulfone [P. Cuatrecasas et al, J. Biol. Chem. 244: 406 (1969)]; and 2,2'-dicarboxy-4,4'-azophenyldiisothiocyanate [H. Fasold, Biochem. Z. 342: 288 (1965)]. The use of such reagents, however, introduces an additional complication. The structural residue contributed by the difunctional linking group introduces another haptenic moiety or antigenic determinant into the immunogen [see M. B. Liu et al, J. Antibiotics 34: 898 (1981); Chem. Abst. 95: 95251t (1981)]. Where the same linking group is used to prepare a labeled conjugate of the hapten for the immunoassay, recognition of the linking group in addition to the distinguishing features of the hapten itself can result in higher affinity of the resulting antibodies for the labeled derivative than for the free analyte and lead to an immunoassay of diminished sensitivity. The need for an inert or benign bridge therefore exists whenever an external label is used as the marker for immunoassays where high sensitivity is required [J. E. T. Corrie et al, J. Endocrinol, 87: 8P (1980)].
The state-of-the-art of preparing antibodies to haptens such as drugs is represented by Weinryb et al, Drug Metabolism Reviews 10: 271(1979); Playfair et al, Br. Med. Bull. 30: 24(1974); Broughton et al, Clin. Chem. 22: 726(1976); and Butler, J. Immunol. Meth. 7: 1(1976) and Pharmacol. Rev. 29(2): 103(1978).
The coupling of di-, tri-, and tetrapeptides to aminomethylated polymers through various coupling routes, including the use of carbonyl diimidazole, is described by A. Orlowska and S. Drabarek, Pol. J. Chem. 54: 2329-36(1980); Chem. Abst. 95: 81506f (1981).